Nucleic acid sample preparation methods and compositions

ABSTRACT

The present invention provides compositions and methods for preparing a nucleic acid library in a multi-purpose buffer (e.g., employing whole genome amplification), where nucleic acid purification is not required between or during steps. In certain embodiments, small amounts of starting nucleic acid (e.g., genomic DNA) are employed and the steps are accomplished in a single container. In some embodiments, the nucleic acid library is subjected to sequencing methodologies or rolling circle amplification.

The present Application claims priority to U.S. Provisional ApplicationSer. No. 61/427,321 filed Dec. 27, 2010, the entirety of which isincorporated by reference herein.

FIELD OF THE INVENTION

The present invention provides compositions and methods for preparing anucleic acid library in a multi-purpose buffer (e.g., employing wholegenome amplification), where nucleic acid purification is not requiredbetween or during steps. In certain embodiments, small amounts ofstarting nucleic acid (e.g., genomic DNA) are employed and the steps areaccomplished in a single container. In some embodiments, the nucleicacid library is subjected to sequencing methodologies or rolling circleamplification.

BACKGROUND

In many fields of research such as genetic diagnosis, cancer research orforensic medicine, the scarcity of genomic DNA can be a severelylimiting factor on the type and quantity of genetic tests that can beperformed on a sample. One approach designed to overcome this problem iswhole genome amplification. The objective is to amplify a limited DNAsample in a non-specific manner in order to generate a new sample thatis indistinguishable from the original but with a higher DNAconcentration. The aim of a typical whole genome amplification techniqueis to amplify a sample up to a microgram level while respecting theoriginal sequence representation.

The first whole genome amplification methods were described in 1992, andwere based on the principles of the polymerase chain reaction. Zhang andcoworkers (Zhang, L., et al. Proc. Natl. Acad. Sci. USA, 1992, 89:5847-5851; herein incorporated by reference) developed the primerextension PCR technique (PEP) and Telenius and collaborators (Teleniuset al., Genomics. 1992, 13(3):718-25; herein incorporated by reference)designed the degenerate oligonucleotide-primed PCR method (DOP-PCR).

PEP involves a high number of PCR cycles, generally using Taq polymeraseand 15 base random primers that anneal at a low stringency temperature.Although the PEP protocol has been improved in different ways, it stillresults in incomplete genome coverage, failing to amplify certainsequences such as repeats. Failure to prime and amplify regionscontaining repeats may lead to incomplete representation of a wholegenome because consistent primer coverage across the length of thegenome provides for optimal representation of the genome. This methodalso has limited efficiency on very small samples (such as singlecells). Moreover, the use of Taq polymerase implies that the maximalproduct length is about 3 kb.

DOP-PCR is a method which generally uses Taq polymerase andsemi-degenerate oligonucleotides (such as CGACTCGAGNNNNNNATGTGG (SEQ IDNO: 12), for example, where N=A, T, C or G) that bind at a low annealingtemperature at approximately one million sites within the human genome.The first cycles are followed by a large number of cycles with a higherannealing temperature, allowing only for the amplification of thefragments that were tagged in the first step. This leads to incompleterepresentation of a whole genome. DOP-PCR generates, like PEP, fragmentsthat are in average 400-500 bp, with a maximum size of 3 kb, althoughfragments up to 10 kb have been reported. On the other hand, as notedfor PEP, a low input of genomic DNA (less than 1 ng) decreases thefidelity and the genome coverage (Kittler et al., Anal. Biochem. 2002,300(2), 237-44).

Multiple displacement amplification (MDA, also known as stranddisplacement amplification; SDA) is a non-PCR-based isothermal methodbased on the annealing of random hexamers to denatured DNA, followed bystrand-displacement synthesis at constant temperature (Blanco et al.,1989, J. Biol. Chem. 264:8935-40, herein incorporated by reference). Ithas been applied to small genomic DNA samples, leading to the synthesisof high molecular weight DNA with limited sequence representation bias(Lizardi et al., Nature Genetics 1998, 19, 225-232; Dean et al., Proc.Natl. Acad. Sci. U.S.A. 2002, 99, 5261-5266; both of which are hereinincorporated by reference). As DNA is synthesized by stranddisplacement, a gradually increasing number of priming events occur,forming a network of hyper-branched DNA structures. The reaction can becatalyzed by the Phi29 DNA polymerase or by the large fragment of theBst DNA polymerase. The Phi29 DNA polymerase possesses a proofreadingactivity resulting in error rates 100 times lower than the Taqpolymerase.

What is needed are whole genome amplification methods that do notrequire nucleic acid purification between or during steps, and/or thatcan be accomplished in a single container.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for preparing anucleic acid library (e.g., a DNA library) in a multi-purpose buffer(e.g., employing whole genome amplification, such as MDA), where nucleicacid purification is not required between or during steps. In certainembodiments, small amounts of starting nucleic acid (e.g., genomic DNA)are employed (e.g., picograms or nanograms) and the steps areaccomplished in a single container. In some embodiments, the nucleicacid library is subjected to sequencing methodologies or rolling circleamplification.

In some embodiments, the present invention provides methods of preparinga nucleic acid library in a multi-purpose buffer, comprising: a) addinga nucleic acid sample (e.g., genomic DNA) to a multi-purpose buffer,wherein the multi-purpose buffer comprises dNTPs and primers; b)contacting the multi-purpose buffer with a plurality of substantiallypurified enzymes, wherein the enzymes have polymerase activity, kinaseactivity, and phosphatase activity, and wherein the contacting is underconditions such that amplified nucleic acid is generated; c) treatingthe multi-purpose buffer (e.g., by mechanical, chemical, or enzymaticmethods) such that sheared amplified nucleic acid is generated; d)treating the multi-purpose buffer to inactivate the polymerase activity;and e) contacting the multi-purpose buffer with a ligase and nucleicacid adapters under conditions such that an adapter-linked nucleic acidlibrary is generated; wherein the above steps are completed in themulti-purpose buffer without nucleic acid purification between or duringsome or all of the steps.

In particular embodiments, the amplified nucleic acid is generated bywhole genome amplification (e.g., MDA). In further embodiments, some orall of the steps (e.g., steps a)-e)) are conducted in a singlecontainer. In other embodiments, the methods further comprise treatingthe multi-purpose buffer containing the adapter-linked nucleic acidlibrary such that proteins and dNTPs are removed from the multi-purposebuffer. In particular embodiments, the treating comprises contacting themulti-purpose buffer with a proteinase and phosphatase, or columnpurifying the multi-purpose buffer. In other embodiments, the adapterscomprise hairpin primers, and wherein the adapter-linked nucleic librarycomprises circular templates.

In further embodiments, the methods further comprise treating themulti-purpose buffer containing the circular templates with at least oneexonuclease enzyme capable of digesting any non-circularized nucleicacid present. In some embodiments, the methods further comprise heatingthe multi-purpose buffer containing the exonuclease enzyme such that theexonuclease is inactivated. In additional embodiments, the adapterscomprise 3′ and/or 5′ blocking groups, and wherein the adapter-linkedlibrary comprises end-blocked templates. In other embodiments, themethods further comprise treating the multi-purpose buffer containingthe end-blocked templates with at least one exonuclease enzyme capableof digesting any non-end-blocked nucleic acid present. In additionalembodiments, the methods further comprise heating the multi-purposebuffer containing the exonuclease enzyme such that the exonuclease isinactivated.

In some embodiments, the multi-purpose buffer further comprises anemulsifier. In certain embodiments, the emulsifier is a polysorbate(e.g., Tween 20, Tween 40, Tween 60, or Tween 80). In other embodiments,the multi-purpose buffer further comprisestris(hydroxymethyl)aminomethane (TRIS). In further embodiments, themulti-purpose buffer further comprises a divalent metal cation. Inadditional embodiments, the multi-purpose buffer further comprises aninorganic salt. In additional embodiments, the inorganic salt isammonium sulfate.

In additional embodiments, the multi-purpose buffer further comprisespolyadenylic acid. In other embodiments, the multi-purpose bufferfurther comprises an alpha-linked disaccharide. In some embodiments, thealpha-linked disaccharide comprises Trehalose. In further embodiments,the multi-purpose buffer further comprises a reducing agent. Inparticular embodiments, the reducing agent further comprisesdithiothreitol (DTT). In certain embodiments, the multi-purpose bufferfurther comprises albumin or an albumin-like protein.

In some embodiments, the methods further comprise, after step c),incubating the multi-purpose buffer such that phosphorylated blunt ends(and/or A-tailed ends) are generated in the sheared amplified nucleicacid. In other embodiments, the genomic DNA is an amount that is between10 pg and 50 ng (e.g., 10 pg to 50 pg, 50 pg to 1 ng, or 1 ng to 50 ng).In some embodiments, the adapter-linked nucleic acid library issubjected to a sequencing methodology or to rolling circleamplification. In certain embodiments, the plurality of substantiallypurified enzymes comprises phi 29 polymerase, Klenow exo-polymerase,polynucleotide kinase, a pyrophosphatase enzyme, or any combinationthereof.

In some embodiments, the present invention provides compositionscomprising at least four (or at least five, or at least six, or at leastseven or at least eight) of the following: a) a buffering agent, b) anemulsifier, c) a divalent metal cation, d) an inorganic salt, e)polyadenylic acid, f) an alpha-linked disaccharide, g) a reducing agent,and h) albumin or an albumin-like protein.

In certain embodiments, the compositions further comprisetris(hydroxymethyl)aminomethane (TRIS). In other embodiments, theemulsifier is a polysorbate. In some embodiments, the polysorbate isselected from the group consisting of: Tween 20, Tween 40, Tween 60, orTween 80. In further embodiments, the inorganic salt is ammoniumsulfate. In additional embodiments, the alpha-linked disaccharidecomprises Trehalose. In some embodiments, the reducing agent comprisesdithiothreitol (DTT).

In further embodiments, the compositions further comprise dNTPs and/orprimers. In other embodiments, the compositions further comprise aplurality of substantially purified enzymes, wherein the enzymes havepolymerase activity, kinase activity, and phosphatase activity. In someembodiments, the plurality of substantially purified enzymes comprises aPhi 29 polymerase, Klenow exo-polymerase, a polynucleotide kinase, apyrophosphatase, a ligase, or any combination thereof.

In some embodiments, the compositions further comprise nucleic acidadapters. In certain embodiments, the adapters comprise hairpin primers.In other embodiments, the compositions further comprise an exonuclease.In certain embodiments, the exonuclease is Exonuclease III orExonuclease VII. In additional embodiments, the compositions furthercomprise random primers. In other embodiments, the random primers aresuitable for use in whole genome amplification methods, such as MDA.

DESCRIPTION OF THE FIGURES

FIG. 1 shows an exemplary embodiment of a single tube library prepprocess in flowchart form. Specific details listed (enzymes/incubationtemps & times, etc.) are merely exemplary and may vary for differenttypes of libraries prepared.

FIG. 2 shows results from Example 1 and shows the ability to amplify nglevels of DNA to ug levels in 30 minutes using phi 29/klenowexo-polymerases in whole genome amplification (WGA). FIG. 2A shows totalyield of amplified product as measured by qPCR, and FIG. 2B shows a gelelectrophoresis analysis of amplified product.

FIG. 3 shows results from Example 1 and shows the ability of sonicationto fragment the WGA DNA in WGA buffer to lengths in the 100s of basepairs.

FIG. 4 shows results from Example 1 and shows the ability of phi29/klenow exo-enzyme mix in WGA buffer to blunt and a-tail DNA using DNAoligos and mass spectrometry for analysis. Results of sample analysis byESI-TOF mass spectrometry using the T5000 system show both before (FIG.4A) and after (FIG. 4B) addition of WGA enzymes.

FIG. 5 shows results from Example 1 and shows the ability of phi29/klenow exo-enzyme mix in WGA buffer to blunt end DNA using DNA oligosand mass spectrometry for analysis. Results of sample analysis byESI-TOF mass spectrometry using the T5000 system show sample analysisboth before (FIG. 5A) and after (FIG. 5B) the addition of WGA enzymes.

FIG. 6 shows results from Example 1 and shows the ability of polynucleotide kinase in WGA buffer to phosphorylate the 5′ ends of DNAusing DNA oligos and mass spectrometry for analysis. FIG. 6 showsanalysis of the sample with the oligos before (FIG. 6A) and after (FIG.6B) addition of polynucleotide kinase.

FIG. 7 shows results from Example 1 and shows the ability to ligate DNAfragments using T4 ligase in WGA buffer using DNA oligos and gelelectrophoresis analysis.

FIG. 8 shows results from Example 1 and shows the ability to ligate DNAfragments using T4 ligase in WGA buffer using DNA oligos and massspectrometry analysis. Sample analysis with (FIG. 8A) and without ligase(FIG. 8B) was conducted with an ESI-TOF mass spectrometer.

FIG. 9 shows results from Example 1 and shows the ability to exonucleasedigest DNA using exonuclease III and exonuclease VII in WGA buffer usingDNA oligos and gel electrophoresis analysis.

FIG. 10 shows results from Example 1 and shows the ability toexonuclease digest DNA using exonuclease III and exonuclease VII in WGAbuffer using DNA oligos and mass spectrometry analysis. This treatedsample (FIG. 10B) and a ligation reaction not treated with theexonucleases (FIG. 10B) were then run on an ESI-TOF mass spectrometerusing the T5000 system.

DETAILED DESCRIPTION

The present invention provides compositions and methods for preparing anucleic acid library in a multi-purpose buffer (e.g., employing wholegenome amplification), where nucleic acid purification is not requiredbetween or during steps. In certain embodiments, small amounts ofstarting nucleic acid (e.g., genomic DNA) are employed and the steps areaccomplished in a single container. In some embodiments, the nucleicacid library is subjected to sequencing methodologies or rolling circleamplification.

Current methods of preparing DNA libraries generally use a physicalmethod for shearing of the DNA such as sonication, nebulization, etc.This is followed by a selection of fragments of the appropriate lengthand then enzymatic steps (including ligation of DNA adapters) to preparethe sample for sequencing. These methods require large amounts ofstarting material (e.g., ugs), significant amounts of time (many hours)and purification of the DNA between each step. The use of a processwhich does not need purification between each of the individual stepsand which contains a whole genome amplification step would allow for asimpler one tube process. This in turn would allow for the creation ofDNA libraries from significantly smaller amounts of starting templatewith a greatly reduced amount of time and effort. Such methods areprovided by the present invention. The present invention provides rapidand simple methods which require no purification between steps, whichcan be conducted in a single tube/container, for the creation of DNAlibraries from small amounts of starting DNA template for use in DNAsequencing or other applications.

In certain embodiments, the present invention uses whole genomeamplification (using enzymes such as phi 29 and klenow exo-polymerases),a physical DNA fragmentation method (such as sonication), an endrepair/a-tailing reaction (using enzymes such as phi 29 polymerase,klenow exo-polymerase and poly-nucleotide kinase), a ligation reactionwith DNA adapters (using enzymes such as T4 ligase) and an exonucleasetreatment (using enzymes such as exonuclease III and exonuclease VII) tocreate a library of DNA templates. In certain embodiments, the presentinvention requires less time (e.g., 30 minutes to 1 hour), less startingmaterial and less manipulation/hands on time to create DNA libraries. Inparticular embodiments, the methods of the present invention areintegrated into an automated microfluidic/Robotic system.

In some embodiments, the resulting DNA libraries are subjected tosequencing technologies. Appropriate adapters, based on the sequencingmethod, are employed to created the DNA library. Exemplary sequencingtechnologies are described below.

Illustrative non-limiting examples of nucleic acid sequencing techniquesinclude, but are not limited to, chain terminator (Sanger) sequencingand dye terminator sequencing, as well as “next generation” sequencingtechniques. Those of ordinary skill in the art will recognize thatbecause RNA is less stable in the cell and more prone to nuclease attackexperimentally RNA is usually reverse transcribed to DNA beforesequencing.

Chain terminator sequencing uses sequence-specific termination of a DNAsynthesis reaction using modified nucleotide substrates. Extension isinitiated at a specific site on the template DNA by using a shortradioactive, or other labeled, oligonucleotide primer complementary tothe template at that region. The oligonucleotide primer is extendedusing a DNA polymerase, standard four deoxynucleotide bases, and a lowconcentration of one chain terminating nucleotide, most commonly adi-deoxynucleotide. This reaction is repeated in four separate tubeswith each of the bases taking turns as the di-deoxynucleotide. Limitedincorporation of the chain terminating nucleotide by the DNA polymeraseresults in a series of related DNA fragments that are terminated only atpositions where that particular di-deoxynucleotide is used. For eachreaction tube, the fragments are size-separated by electrophoresis in aslab polyacrylamide gel or a capillary tube filled with a viscouspolymer. The sequence is determined by reading which lane produces avisualized mark from the labeled primer as you scan from the top of thegel to the bottom.

Dye terminator sequencing alternatively labels the terminators. Completesequencing can be performed in a single reaction by labeling each of thedi-deoxynucleotide chain-terminators with a separate fluorescent dye,which fluoresces at a different wavelength.

A set of methods referred to as “next-generation sequencing” techniqueshave emerged as alternatives to Sanger and dye-terminator sequencingmethods (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLeanet al., Nature Rev. Microbiol., 7: 287-296; each herein incorporated byreference in their entirety). Next-generation sequencing (NGS) methodsshare the common feature of massively parallel, high-throughputstrategies, with the goal of lower costs in comparison to oldersequencing methods. NGS methods can be broadly divided into those thatrequire template amplification and those that do not.Amplification-requiring methods include pyrosequencing commercialized byRoche as the 454 technology platforms (e.g., GS 20 and GS FLX), theSolexa platform commercialized by Illumina, and the SupportedOligonucleotide Ligation and Detection (SOLiD) platform commercializedby Applied Biosystems. Non-amplification approaches, also known assingle-molecule sequencing, are exemplified by the HeliScope platformcommercialized by Helicos BioSciences, and emerging platformscommercialized by VisiGen, Oxford Nanopore Technologies Ltd., andPacific Biosciences, respectively.

In pyrosequencing (Voelkerding et al., Clinical Chem., 55: 641-658,2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No.6,210,891; U.S. Pat. No. 6,258,568; each herein incorporated byreference in its entirety), template DNA is fragmented, end-repaired,ligated to adaptors, and clonally amplified in-situ by capturing singletemplate molecules with beads bearing oligonucleotides complementary tothe adaptors. Each bead bearing a single template type iscompartmentalized into a water-in-oil microvesicle, and the template isclonally amplified using a technique referred to as emulsion PCR. Theemulsion is disrupted after amplification and beads are deposited intoindividual wells of a picotitre plate functioning as a flow cell duringthe sequencing reactions. Ordered, iterative introduction of each of thefour dNTP reagents occurs in the flow cell in the presence of sequencingenzymes and luminescent reporter such as luciferase. In the event thatan appropriate dNTP is added to the 3′ end of the sequencing primer, theresulting production of ATP causes a burst of luminescence within thewell, which is recorded using a CCD camera. It is possible to achieveread lengths greater than or equal to 400 bases, and 1×10⁶ sequencereads can be achieved, resulting in up to 500 million base pairs (Mb) ofsequence.

In the Solexa/Illumina platform (Voelkerding et al., Clinical Chem., 55:641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S.Pat. No. 6,833,246; U.S. Pat. No. 7,115,400; U.S. Pat. No. 6,969,488;each herein incorporated by reference in its entirety), sequencing dataare produced in the form of shorter-length reads. In this method,single-stranded fragmented DNA is end-repaired to generate5′-phosphorylated blunt ends, followed by Klenow-mediated addition of asingle A base to the 3′ end of the fragments. A-addition facilitatesaddition of T-overhang adaptor oligonucleotides, which are subsequentlyused to capture the template-adaptor molecules on the surface of a flowcell that is studded with oligonucleotide anchors. The anchor is used asa PCR primer, but because of the length of the template and itsproximity to other nearby anchor oligonucleotides, extension by PCRresults in the “arching over” of the molecule to hybridize with anadjacent anchor oligonucleotide to form a bridge structure on thesurface of the flow cell. These loops of DNA are denatured and cleaved.Forward strands are then sequenced with reversible dye terminators. Thesequence of incorporated nucleotides is determined by detection ofpost-incorporation fluorescence, with each fluor and block removed priorto the next cycle of dNTP addition. Sequence read length ranges from 36nucleotides to over 50 nucleotides, with overall output exceeding 1billion nucleotide pairs per analytical run.

Sequencing nucleic acid molecules using SOLiD technology (Voelkerding etal., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev.Microbiol., 7: 287-296; U.S. Pat. No. 5,912,148; U.S. Pat. No.6,130,073; each herein incorporated by reference in their entirety) alsoinvolves fragmentation of the template, ligation to oligonucleotideadaptors, attachment to beads, and clonal amplification by emulsion PCR.Following this, beads bearing template are immobilized on a derivatizedsurface of a glass flow-cell, and a primer complementary to the adaptoroligonucleotide is annealed. However, rather than utilizing this primerfor 3′ extension, it is instead used to provide a 5′ phosphate group forligation to interrogation probes containing two probe-specific basesfollowed by 6 degenerate bases and one of four fluorescent labels. Inthe SOLiD system, interrogation probes have 16 possible combinations ofthe two bases at the 3′ end of each probe, and one of four fluors at the5′ end. Fluor color and thus identity of each probe corresponds tospecified color-space coding schemes. Multiple rounds (usually 7) ofprobe annealing, ligation, and fluor detection are followed bydenaturation, and then a second round of sequencing using a primer thatis offset by one base relative to the initial primer. In this manner,the template sequence can be computationally re-constructed, andtemplate bases are interrogated twice, resulting in increased accuracy.Sequence read length averages 35 nucleotides, and overall output exceeds4 billion bases per sequencing run.

In certain embodiments, nanopore sequencing in employed (see, e.g.,Astier et al., J Am Chem Soc. 2006 Feb. 8; 128(5):1705-10, hereinincorporated by reference). The theory behind nanopore sequencing has todo with what occurs when the nanopore is immersed in a conducting fluidand a potential (voltage) is applied across it: under these conditions aslight electric current due to conduction of ions through the nanoporecan be observed, and the amount of current is exceedingly sensitive tothe size of the nanopore. If DNA molecules pass (or part of the DNAmolecule passes) through the nanopore, this can create a change in themagnitude of the current through the nanopore, thereby allowing thesequences of the DNA molecule to be determined.

HeliScope by Helicos BioSciences (Voelkerding et al., Clinical Chem.,55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296;U.S. Pat. No. 7,169,560; U.S. Pat. No. 7,282,337; U.S. Pat. No.7,482,120; U.S. Pat. No. 7,501,245; U.S. Pat. No. 6,818,395; U.S. Pat.No. 6,911,345; U.S. Pat. No. 7,501,245; each herein incorporated byreference in their entirety) is the first commercialized single-moleculesequencing platform. This method does not require clonal amplification.Template DNA is fragmented and polyadenylated at the 3′ end, with thefinal adenosine bearing a fluorescent label. Denatured polyadenylatedtemplate fragments are ligated to poly(dT) oligonucleotides on thesurface of a flow cell. Initial physical locations of captured templatemolecules are recorded by a CCD camera, and then label is cleaved andwashed away. Sequencing is achieved by addition of polymerase and serialaddition of fluorescently-labeled dNTP reagents. Incorporation eventsresult in fluor signal corresponding to the dNTP, and signal is capturedby a CCD camera before each round of dNTP addition. Sequence read lengthranges from 25-50 nucleotides, with overall output exceeding 1 billionnucleotide pairs per analytical run.

Another exemplary nucleic acid sequencing approach developed by StratosGenomics, Inc. that is also optionally adapted for use with the presentinvention involves the use of Xpandomers. This sequencing processtypically includes providing a daughter strand produced by atemplate-directed synthesis. The daughter strand generally includes aplurality of subunits coupled in a sequence corresponding to acontiguous nucleotide sequence of all or a portion of a target nucleicacid in which the individual subunits comprise a tether, at least oneprobe or nucleobase residue, and at least one selectively cleavablebond. The selectively cleavable bond(s) is/are cleaved to yield anXpandomer of a length longer than the plurality of the subunits of thedaughter strand. The Xpandomer typically includes the tethers andreporter elements for parsing genetic information in a sequencecorresponding to the contiguous nucleotide sequence of all or a portionof the target nucleic acid. Reporter elements of the Xpandomer are thendetected. Additional details relating to Xpandomer-based approaches aredescribed in, for example, U.S. Patent Publication No. 20090035777,entitled “HIGH THROUGHPUT NUCLEIC ACID SEQUENCING BY EXPANSION,” thatwas filed Jun. 19, 2008, which is incorporated herein in its entirety.

Other emerging single molecule sequencing methods include real-timesequencing by synthesis using a VisiGen platform (Voelkerding et al.,Clinical Chem., 55: 641-658, 2009; U.S. Pat. No. 7,329,492; U.S. patentapplication Ser. No. 11/671,956; U.S. patent application Ser. No.11/781,166; each herein incorporated by reference in their entirety) inwhich immobilized, primed DNA template is subjected to strand extensionusing a fluorescently-modified polymerase and florescent acceptormolecules, resulting in detectible fluorescence resonance energytransfer (FRET) upon nucleotide addition.

Another real-time single molecule sequencing system developed by PacificBiosciences (Voelkerding et al., Clinical Chem., 55: 641-658, 2009;MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No.7,170,050; U.S. Pat. No. 7,302,146; U.S. Pat. No. 7,313,308; U.S. Pat.No. 7,476,503; all of which are herein incorporated by reference)utilizes reaction wells 50-100 nm in diameter and encompassing areaction volume of approximately 20 zeptoliters (10×10⁻²¹ L). Sequencingreactions are performed using immobilized template, modified phi29 DNApolymerase, and high local concentrations of fluorescently labeleddNTPs. High local concentrations and continuous reaction conditionsallow incorporation events to be captured in real time by fluor signaldetection using laser excitation, an optical waveguide, and a CCDcamera.

In certain embodiments, the single molecule real time (SMRT) DNAsequencing methods using zero-mode waveguides (ZMWs) developed byPacific Biosciences, or similar methods, are employed. With thistechnology, DNA sequencing is performed on SMRT chips, each containingthousands of zero-mode waveguides (ZMWs). A ZMW is a hole, tens ofnanometers in diameter, fabricated in a 100 nm metal film deposited on asilicon dioxide substrate. Each ZMW becomes a nanophotonic visualizationchamber providing a detection volume of just 20 zeptoliters (10⁻²¹liters). At this volume, the activity of a single molecule can bedetected amongst a background of thousands of labeled nucleotides.

The ZMW provides a window for watching DNA polymerase as it performssequencing by synthesis. Within each chamber, a single DNA polymerasemolecule is attached to the bottom surface such that it permanentlyresides within the detection volume. Phospholinked nucleotides, eachtype labeled with a different colored fluorophore, are then introducedinto the reaction solution at high concentrations which promote enzymespeed, accuracy, and processivity. Due to the small size of the ZMW,even at these high, biologically relevant concentrations, the detectionvolume is occupied by nucleotides only a small fraction of the time. Inaddition, visits to the detection volume are fast, lasting only a fewmicroseconds, due to the very small distance that diffusion has to carrythe nucleotides. The result is a very low background.

Processes and systems for such real time sequencing that may be adaptedfor use with the invention are described in, for example, U.S. Pat. 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monitoring multiple optical signals from a single source”, filedAug. 11, 2005 by Lundquist et al., and Korlach et aI. (2008) “Selectivealuminum passivation for targeted immobilization of single DNApolymerase molecules in zero-mode waveguide nanostructures” Proc. Nat'I.Acad. Sci. U.S.A. 105(4): 11761181—all of which are herein incorporatedby reference in their entireties.

EXAMPLES Example 1 Single-Tube DNA Library Generation

This Example describes an exemplary single-tube method for generatingDNA libraries from small amount of starting material that does notrequire DNA purification between steps and which employs a universalbuffer. The method is generally outlined in FIG. 1. Briefly, the processstarts with an aqueous samples which is then subjected to alysis/nucleic acid extraction protocol and eluted in a universal wholegenome amplification (WGA) buffer. The composition of the buffer isshown in Table 1:

TABLE 1 Tris Ph 7.5 50.00 mM MgCl2 9.00 mM (NH4)2SO4 7.50 mM SonicatedPoly Adenylic acid 1.00 Ng/ul Trehalose 0.60M DNTP mix 100 mM (25 mMeach) Bioline 2.00 mM DTT 4.00 mM Primers 50.00 uM BSA 0.23 ug/ulTween-40 1.00%The sample is then heated at 95 C. for 1 minute to denature the genomicDNA and allow the short random primers to bind. After cooling to anappropriate temperature (e.g., ˜37 C.) the WGA enzyme mix is added,which includes phi 29 polymerase, klenow exo-polymerase, polynucleotidekinase and pyrophosphatase, and incubated for 30 minutes at 37 C. toamplify the genomic material.

The sample is then physically sheared (e.g., by sonication) and allowedto incubate another 30 minutes at 30 C. to polish the ends of the nowsheared molecules. After incubation at an elevated temperature toinactivate the polymerase (e.g., 75 C. for 10 minutes) DNA linkers areadded (in this Example, hairpin oligos as described below) as well as T4ligase and ATP. The reaction is allowed to incubate at 25 C. for anappropriate amount of time (blunt ends are done in ˜15 minutes, A-tailedend reactions take longer.) In this Example, single stranded circularDNA molecules are generated. As such, exonucleases are then added toremove any non circularized DNA present with an incubation at 37 C. Itis noted that if this Example were creating non-circularized templatesfor different sequencing technologies, one could change exonucleases andinclude some 5′ and/or 3′ blocking agents on the adapters to achieve thesame result. The exonucleases are then heat inactivated at 95 C and thesample treated with further clean up reagents, including Pro-K to removeenzyme/protein components and a phosphatase to remove unused dNTPs(although other clean up procedures such as those with resin could alsobe used.) After an appropriate incubation time and temperature andelevated temperature to inactivate the clean up enzymes the reactionsproceeds through a final clean up/size selection process (e.g., such asbind-elute and flow through resins).

FIG. 2 shows the yield that can be obtained with WGA starting with 1 ngof Klebsiella pneumoniae (Kp) genomic DNA using the methods describedabove. By Kp specific DNA the yield is over 2.5 ug (2500× foldamplification) with gel electrophoresis showing the typical smearpattern seen with WGA reactions and an appropriate amount of total DNA.

The materials and methods used for generating the data in FIG. 2 are asfollows. One (1) ng K. pneumoniae (Kp) purified genomic DNA serves asstarting material. The buffer in Table 1, 100 units Phi 29, and 40 unitsKlenow exo- are employed for WGA using the following reactionconditions: 100 ul total volume, heat to 95 C. for 1 m prior to enzymeaddition, cool samples to 4 C., then after adding enzyme and mixing, 37C. for 30 min, then 75 C. for 10 min. FIG. 2A shows total yield ofamplified product as measured by qPCR, and FIG. 2B shows a gelelectrophoresis analysis of amplified product using 1% agarose, ethidiumbromide UV light visualization.

Whole genome DNA (20 ul) as prepared in FIG. 2 was sonicated in a thinwall PCR tube floating in a 4 C. water bath using a cup horn sonicator(Misonix 3000, power level 10 (full power ˜200 w)) for the appropriateamount of time. 10 ul aliquots of these reactions were then run on 1%EtBr/agarose gel for 45 minutes at 100V and a UV light source. FIG. 3shows the effect of different amounts of sonication time on the size ofWGA DNA in A9 buffer. After 5 minutes most of the DNA is in the 100's ofby range which is a reasonable range for a number of sequencingtechnologies (although if smaller pieces are needed further sonicationcan reduce the size further.)

Next, “before rxn” oligos (shown below, final concentration of 1 μMeach) were hybridized to each other and mixed with buffer in Table 1 and100 units phi 29 and 40 units klenow exo-. This reaction was thenincubated for 30 min at 30 C. followed by 75 C. for 10 minutes. Thesample was then analyzed by ESI-TOF mass spectrometry using the T5000system both before (FIG. 4A) and after (FIG. 4B) addition of WGAenzymes. The sequences of end polished oligos are shown below with anaddition untemplated A (majority product)

Before Rxn

Top strand: (SEQ ID NO: 1) CATGCGGATGCAGAGGAGGACGACTCTGATGTCTBottom strand: (SEQ ID NO: 2)GCAATGAAGACATCAGAGTCGTCCTCCTCTGCATCCGCATGTGTAfter Rxn (with +a Shown)

Top strand: (SEQ ID NO: 3) CATGCGGATGCAGAGGAGGACGACTCTGATGTCTTCATTGCABottom strand: (SEQ ID NO: 4) GCAATGAAGACATCAGAGTCGTCCTCCTCTGCATCCGCATGA

In FIG. 4 it is shown that the enzymes used for WGA in the A9 bufferwill end polish and A-tail DNA oligos used to model sheared DNA whenincubated at 30 C. for 30 minutes. In this portion of the Example, a setof oligos was used that when hybridized have both a 5′ and 3′ overhangallowing for the WGA enzymes to chew back the 3′ overhang and fill inopposite the 5′ overhang giving blunt end DNA molecules which can thenhave an untemplated A-added. After the incubation at 30 C. for 30 min,the samples were heated at 75 C. for 10 minutes and analyzed using anESI-TOF mass spectrometry (T5000 system.)

Next, “before rxn” oligos (shown below, final concentration of 1 μMeach) were hybridized to each other and mixed with buffer in Table 1above and 100 units phi 29 and 40 units klenow exo-. This reaction wasthen incubated for 5 min at 37 C. followed by 75 C. for 10 minutes. Thesample was then analyzed by ESI-TOF mass spectrometry using the T5000system showing sample analysis both before (FIG. 5A) and after (FIG. 5B)the addition of WGA enzymes. The sequences of end polished oligosdepicted in the right portion of the figure are shown below with bluntends (majority product).

Before Rxn

Top strand: (SEQ ID NO: 1) CATGCGGATGCAGAGGAGGACGACTCTGATGTCTBottom strand: (SEQ ID NO: 2)GCAATGAAGACATCAGAGTCGTCCTCCTCTGCATCCGCATGTGTAfter Rxn (with +A Shown)

Top strand: (SEQ ID NO: 5) CATGCGGATGCAGAGGAGGACGACTCTGATGTCTTCATTGCBottom strand: (SEQ ID NO: 6) GCAATGAAGACATCAGAGTCGTCCTCCTCTGCATCCGCATG

In FIG. 5, its shown that the enzymes used for WGA in the buffer fromTable 1 will end polish but not A-tail DNA oligos used to model shearedDNA when incubated at 37 C. for 5 minutes. Specifically, when the aboveset of oligos were used which, when hybridized, have both a 5′ and 3′overhang allowing for the WGA enzymes to chew back the 3′ overhang andfill in opposite the 5′ overhang giving blunt end DNA molecules. Afterthe incubation at 37 C. for 5 min, the samples were heated at 75 C. for10 minutes and analyzed using an ESI-TOF mass spectrometry (T5000system.)

Next, two complementary oligos were hybridized to each other giving ablunt end duplex with no 5′ or 3′ phosphates. This duplex was mixed withthe buffer from Table 1 and 5 units polynucleotide kinase and incubatedfor 30 minutes at 30 C. followed by 75 C. for 10 min. The sample wasthen analyzed by ESI-TOF mass spectrometry using the T5000 system.

(SEQ ID NO: 7) Oligo#1 5' TGCGGATGCAGAGGAGGATGACTCTGATGTCT(SEQ ID NO: 8) Oligo#2 5' AGACATCAGAGTCATCCTCCTCTGCATCCGCA

In FIG. 6 shows that polynucleotide kinase will phosphorylate DNA in thebuffer from Table 1. Specifically, the above set of oligos, which whenhybridized, have blunt ends and no 5′ phosphate were employed. Afterincubation with polynucleotide kinase at 37 C. for 30 minutes, thereactions were heated to 75 C. for 10 min and analyzed using ESI-TOFmass spectrometry (T5000 system.). FIG. 6 shows analysis of the samplewith the oligos before (FIG. 6A) and after (FIG. 6B) addition ofpolynucleotide kinase.

Next, two complementary oligos each with a 5′ overhang were hybridizedtogether. A hairpin oligo with a complementary 5′ overhang was alsohybridized separately. These oligos were then mixed in the buffer fromTable 1 with 1000 cohesive end ligation units of T4 DNA ligase. Thisreaction was then incubated for the appropriate amount of time (30, 60,120 min) at 16 C. followed by 75 C. for 10 minutes. The samples(including controls of only hairpin, only insert and hairpin+insert butwith no ligase) were then run on a 1% agarose gel and visualized usingethidium bromide and a uv light source.

Insert Oligos:

(SEQ ID NO: 9) 5'-P-GAAGCATGCGGATGCAGAGGAGGACGACTCTGATGTCTTCATTGC(SEQ ID NO: 10) 5'-P-GAAGGCAATGAAGACATCAGAGTCGTCCTCCTCTGCATCCGCATG

Hairpin Oligo

(SEQ ID NO: 11) 5'-P-CTTC TCTCTCTCttttcctcctcctccgttgttgttgttGAGAG AGAComplementary 5′ overhangs are in bold, complementary stem of hairpinstructure is underlined, lowercase bases of hairpin indicate unpairedbases

FIG. 7 shows that by gel electrophoresis, one can perform ligationreactions in the buffer in Table 1 using T4 DNA ligase and ATP.Specifically, the above set of oligos which, when hybridized, have a 5′“sticky end” on both ends of the duplex DNA molecule and a hairpin oligowhich has a complementary 5′ “sticky end” overhang. Ligations wereperformed at 16 C. in the buffer in Table 1 with ATP added. Foranalysis, gel electrophoresis was used and it showed that after 30minutes the reaction was complete giving mostly a product that migratesat ˜120 bp, a minor product which migrates at ˜75 bp and no “insert”starting material (which migrates at ˜50 bp.) (Note: the hairpin oligois not visible on the gel despite its high concentration due to thesignificant single stranded portion of the oligo which allow for minimalintercalation by ethidium bromide.)

Next, the same two complementary oligos each with a 5′ overhang werehybridized together (SEQs 9 and 10). A hairpin oligo with acomplementary 5′ overhang was also hybridized separately (SEQ ID NO:11).These oligos were then mixed in the buffer in Table 1 with 1000 cohesiveend ligation units of T4 DNA ligase. This reaction was then incubatedfor the appropriate amount of time 30 minutes at 16 C. followed by 75 C.for 10 minutes. The samples (including a control of hairpin+insert butwith no ligase) were then run on an ESI-TOF mass spectrometer using theT5000 system.

FIG. 8 shows that by mass spectral analysis one can perform ligationreactions in the buffer in Table 1 using T4 DNA ligase and ATP. The setof oligos show above were used which when hybridized have a 5′ “stickyend” on both ends of the duplex DNA molecule and a hairpin oligo whichhas a complementary 5′ “sticky end” overhang. Ligations were performedat 16 C. for 30 min in the buffer in Table 1 with ATP added. Foranalyis, a mixture of hairpin “insert” oligos and “hairpin” oligos with(FIG. 8A) or without the addition of ligase (FIG. 8B) were analyzedusing ESI-TOF mass spectrometry. This showed that without ligase, onlystarting materials were visualized, but with ligase, no insert oligoswere visualized and instead the presence of a high molecular weightproduct which corresponds to an insert duplex molecule with a hairpinoligo ligated on either end (hairpin is still observed in +ligasereaction because it was at 10× the insert starting concentration.)

Next, two complementary oligos each with a 5′ overhang were hybridizedtogether (SEQs 9 and 10). A hairpin oligo with a complementary 5′overhang was also hybridized separately (SEQ ID NO:11). These oligoswere then mixed in the buffer from Table 1 with 1000 cohesive endligation units of T4 DNA ligase. This reaction was then incubated forthe appropriate amount of time 30 minutes at 16 C. followed by 75 C. for10 minutes. The reaction was then mixed with exonuclease III andexonuclease VII and incubated for 30 minutes at 37 C. This sample andcontrols of only insert, only hairpin, and a ligation reaction nottreated with the exonucleases was run on a 1% agarose gel and visualizedby ethidium bromide and a uv light source.

FIG. 9 shows that exonucleases (specifically Exo II and Exo VII) arefunctional in the buffer in Table 1 and can be used to removenon-circular DNA products from the reaction. These reactions usedligation reactions as run in FIGS. 8 and 9 and subjected them toexonuclease III and exonuclease VII for 30 minutes at 37 C. The sampleswere then analyzed using gel electrophoresis showing the removal ofnon-circular products and the retention of circular products.

Next, the same two complementary oligos each with a 5′ overhang werehybridized together (SEQs 9 and 10). A hairpin oligo with acomplementary 5′ overhang was also hybridized separately (SEQ ID NO:11).These oligos were then mixed in the buffer from Table 1 with 1000cohesive end ligation units of T4 DNA ligase. This reaction was thenincubated for the appropriate amount of time 30 minutes at 16 C.followed by 75 C. for 10 minutes. The reaction was then mixed withexonuclease III and exonuclease VII and incubated for 30 minutes at 37C. This sample (FIG. 10B) and a ligation reaction not treated with theexonucleases (FIG. 10B) were then run on an ESI-TOF mass spectrometerusing the T5000 system.

FIG. 10 shows that exonucleases (specifically Exo II and Exo VII) arefunctional in the buffer from Table 1 and can be used to removenon-circular DNA products from the reaction. These reactions usedligation reactions as run in FIGS. 8 and 9 and subjected them toexonuclease III and exonuclease VII for 30 minutes at 37 C. The sampleswere then analyzed using ESI-TOF mass spectrometry showing the removalof non-circular products and the retention of circular products.

All publications and patents mentioned in the present application areherein incorporated by reference. Various modification and variation ofthe described methods and compositions of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention that are obvious to those skilledin the relevant fields are intended to be within the scope of thefollowing claims.

1. A method of preparing a nucleic acid library in a multi-purposebuffer, comprising: a) combining a nucleic acid sample with amulti-purpose buffer, wherein said multi-purpose buffer comprises dNTPsand primers; b) contacting said multi-purpose buffer with a plurality ofsubstantially purified enzymes, wherein said enzymes have polymeraseactivity, kinase activity, and phosphatase activity, and wherein saidcontacting is under conditions such that amplified nucleic acid isgenerated; c) treating said multi-purpose buffer such that shearedamplified nucleic acid is generated; d) treating said multi-purposebuffer to inactivate said polymerase activity; and e) contacting saidmulti-purpose buffer with a ligase and nucleic acid adapters underconditions such that an adapter-linked nucleic acid library isgenerated; wherein the above steps are completed in said multi-purposebuffer without nucleic acid purification.
 2. The method of claim 1,wherein said nucleic acid sample comprises genomic DNA, and wherein theamplified nucleic acid is generated by whole genome amplification. 3.The method of claim 1, wherein said steps a)-e) are conducted in asingle container.
 4. The method of claim 1, further comprising treatingsaid multi-purpose buffer containing said adapter-linked nucleic acidlibrary such that proteins and dNTPs are removed from said multi-purposebuffer.
 5. The method of claim 1, wherein said adapters comprise hairpinprimers, and wherein said adapter-linked nucleic library comprisescircular templates.
 6. The method of claim 5, further comprisingtreating said multi-purpose buffer containing said circular templateswith at least one exonuclease enzyme capable of digesting anynon-circularized nucleic acid present.
 7. The method of claim 6, furthercomprising heating said multi-purpose buffer containing said exonucleaseenzyme such that said exonuclease is inactivated.
 8. The method of claim1, wherein said multi-purpose buffer further comprises an emulsifier. 9.The method of claim 1, wherein said multi-purpose buffer furthercomprises tris(hydroxymethyl)aminomethane (TRIS).
 10. The method ofclaim 1, wherein said multi-purpose buffer further comprises a divalentmetal cation.
 11. The method of claim 1, wherein said multi-purposebuffer further comprises an inorganic salt.
 12. The method of claim 1,wherein said multi-purpose buffer further comprises polyadenylic acid.13. The method of claim 1, wherein said multi-purpose buffer furthercomprises an alpha-linked disaccharide.
 14. The method of claim 1,wherein said multi-purpose buffer further comprises a reducing agent.15. The method of claim 1, wherein said multi-purpose buffer furthercomprises albumin or albumin-like protein.
 16. The method of claim 1,wherein said nucleic acid sample is an amount that is between 10 pg and50 ng.
 17. A composition comprising: a) a buffering agent, b) anemulsifier, c) a divalent metal cation, d) an inorganic salt, e)polyadenylic acid, f) an alpha-linked disaccharide, g) a reducing agent,and h) albumin or an albumin-like protein.
 18. The composition of claim17, further comprising tris(hydroxymethyl)aminomethane (TRIS).
 19. Thecomposition of claim 17, wherein said emulsifier is a polysorbate. 20.The composition of claim 19, wherein said polysorbate is selected fromthe group consisting of: Tween 20, Tween 40, Tween 60, or Tween
 80. 21.The composition of claim 17, wherein said inorganic salt is ammoniumsulfate.
 22. The composition of claim 17, wherein said alpha-linkeddisaccharide comprises Trehalose.
 23. The composition of claim 17,wherein said reducing agent comprises dithiothreitol (DTT).
 24. Thecomposition of claim 17, wherein said composition further comprisesdNTPs and primers.
 25. The composition of claim 17, further comprising aplurality of substantially purified enzymes, wherein said enzymes havepolymerase activity, kinase activity, and phosphatase activity.